PURPOSE To prospectively discriminate between normal prostate tissue, prostatitis and prostate cancer, based on apparent diffusion coefficient (ADC)-values, using MR-guided biopsy specimens as the standard of reference. METHOD AND MATERIALS The requirement to obtain institutional review board approval was waived. MR-guided biopsies were performed in 130 consecutive patients with cancer suspicious regions (CSRs) on multi-parametric MR imaging at 3T. Exclusion criteria were patients with suspicion of prostate cancer recurrence after therapy, and biopsy specimens which could not be categorized within the following histopathological groups: normal prostate tissue, prostatitis and prostate cancer. During the biopsy procedure, an axial diffusion-weighted sequence was acquired with construction of ADC maps (TR/TE, 2000/67 ms; section thickness of 4 mm; in-plane resolution, 1.8 x 1.8 mm and b-values of 0, 100, 500 and 800 s/mm2). To verify the biopsy location, a confirmation scan with the needle left in situ was acquired. This confirmation scan was projected on the calculated ADC map in order to draw a region-of-interest (ROI) on the ADC map, representing the biopsied CSR. The obtained ADC-values of this ROI were compared with the histological outcomes of the biopsy specimens. A one-way ANOVA with post-hoc comparison was used to test for ADC-value differences among the three histological groups. Differences were considered to be significant at p<.05. RESULTS In total 85 out of 130 patients were included in this study. The median ADC-values differed significantly (ANOVA, p<0.001) across normal prostate tissue (1.05x10-3 mm2/s, SD?0.17), prostatitis (1.13x10-3 mm2/s, SD?0.19) and prostate cancer (0.86x10-3 mm2/s, SD?0.13). Bonferroni post-hoc comparisons of the three groups showed that there is a statistically significant difference in median ADC-values of prostate tissue with prostatitis and prostate cancer in the peripheral zone (p<0.001) and the central gland (p=0.03). Furthermore, a statistically significant difference was found in median ADC-values of normal prostate tissue and prostate cancer in the peripheral zone (p<0.001) and the central gland (p<0.001). CONCLUSION Median ADC-values can prospectively discriminate between prostate cancer and normal prostate tissue or prostatitis. CLINICAL RELEVANCE/APPLICATION Fast non-invasive prediction of histological classification with diffusion weighted imaging in the prostate may improve patient management.